Method of detecting or quantitating endogenous wheat dna and method of determining contamination rate of genetically modified wheat in test sample

ABSTRACT

An object of the present invention is to discover an endogenous wheat sequence satisfying the conditions of: a) it is universally present in varieties of wheat, b) the amount present (detected amount) is not affected depending on the wheat variety, c) even if other grains are present, only wheat can be detected without cross-reactivity, and d) it is amplified quantitatively by the PCR reaction. A further object of the present invention is to provide a method of accurately detecting and quantitating endogenous wheat DNA in a test sample by the polymerase chain reaction. The present invention provides a kit for detecting or quantitating an endogenous wheat DNA sequence in a test sample by the polymerase chain reaction, the kit comprising at least one primer pair capable of amplifying the endogenous wheat DNA sequence.

BACKGROUND

The present invention relates to a method of detecting or quantitatingendogenous wheat DNA in a test sample, and more particularly to a methodof detecting or quantitating endogenous wheat DNA to be used whendetermining the contamination rate of genetically modified wheatcontained in a food material or processed food.

In Japan more than 50 varieties of genetically modified crops(hereinafter, “genetically modified organism” or “GMO”) including maize,soybeans, and potatoes have undergone a safety assessment and have beenapproved for import and sale. Accordingly, a food product containing aGMO must be labeled based on the “Labeling Standard for GeneticallyModified Foods issued by the Ministry of Agriculture, Forestry andFisheries that was established in accordance with Article 7, paragraph 1of the Quality Labeling Standard for Processed Foods and on Article 7,paragraph 1 of the Quality Labeling Standard for Fresh Foods”(Notification No. 517 of the Ministry of Agriculture, Forestry andFisheries, 31 Mar. 2000), and on the “Enforcement of MinisterialOrdinance amending portions of Ministerial Ordinances on the FoodSanitation Law Enforcement Regulations and Concerning CompositionalStandards, etc. for Milk and Milk Products” (Notice No. 79 of the FoodSanitation Department, Pharmaceutical and Medical Safety Bureau,Ministry of Health, Labor and Welfare 15 Mar. 2001).

In foreign countries, however, GMO crops may sometimes be cultivatedtogether with non-GMO crops once the safety evaluation has beencompleted, and contamination may occur after harvest during thedistribution process. The makers of food products and the like oftensubcontract the manufacturing of processed foods to manufacturingplants, and even though the contract may stipulate to use a non-GMOmaterial, small amounts of a GMO may contaminate processed foods if aGMO is also used in the same plant. Therefore, to comply with thelabeling obligations the makers of food products must inspect andanalyze the finished processed food products to verify that they are notcontaminated with a GMO.

Test methods for detecting a GMO in a test sample of a processed food,the raw material thereof, etc., include the detection of recombinant DNAby the polymerase chain reaction (hereinafter, “PCR”) and the detectionof a recombinant protein by ELISA. In the case of processed foods,however, proteins often become denatured by heat and pressure, and theycannot be detected accurately by ELISA. Therefore, detection by PCR iscommonly used.

Methods of laboratory analysis include the methods described in JASAnalytical Test Handbook, “Genetically Modified Food Test and AnalysisManual for Individual Products, Revised Second Edition (Non-PatentDocument 1)” and those described in “Testing for Foods Produced byRecombinant DNA Techniques (Partially Revised)” (Notice No. 0618002 ofthe Food Sanitation Department, Ministry of Health, Labor and Welfare,Jun. 18, 2003). These documents describe that in the testing andanalysis of a GMO it is necessary to perform PCR using a primer pairrecognizing the endogenous DNA of each agricultural product and toverify that a PCR product of the predicted length is obtained forverification that DNA extracted from the test sample can be amplified byPCR. When quantitating a GMO contained in a test sample, a method isused of measuring the contamination rate of the modified crop based onthe ratio of recombinant DNA to endogenous DNA that is always present inthat crop.

In the case of maize for example, primer pairs have been developed thatrecognize each of the 5 strains of approved GMO varieties, along with aprimer pair that recognizes the SSIIB gene region as an endogenous maizeDNA (Non-patent document 1).

In “Testing for Foods Produced by Recombinant DNA Techniques (PartiallyRevised)” (Notice No. 1113001 of the Food Sanitation Department,Ministry of Health, Labor and Welfare, 13 Nov. 2003), during the processof performing quantitative PCR, the amplification products amplified byspecific primer pairs targeting endogenous DNA and recombinant DNA ofmaize or soybean are ligated to a plasmid and used as a standardreference material. By performing PCR using this standard referencematerial, the ratio of the number of copies of recombinant DNA to thenumber of copies of endogenous DNA can be accurately determined in atest sample by fixed-time, quantitative PCR.

When there are a plurality of GMO strains, as in the case of maize, oneparticularly useful technique is to utilize a common standard referencematerial to measure the contamination rate of each strain, which can bedone by using a standard reference material having endogenous DNA andDNAs specific to each strain incorporated into a single circular DNAmolecule.

It is generally difficult to obtain genes specific to each strain, butonce replicable DNA incorporating those genes has been prepared, it ispossible to stably provide strain-specific DNA by replication thereof.

While no genetically modified products of common wheat have yet passedsafety assessment, they are expected to appear on the market in the nearfuture. Consequently, methods for detecting and quantitating endogenouswheat DNA and PCR primer pairs for use in such methods need to bedeveloped to prepare for the distribution of GMO wheat.

The forms of genes found in wheat have diverse variations compared withother grains. That is because hexaploid, tetraploid, and diploidgenotypes occur depending on the variety of wheat. For example, generalcommon wheat is hexaploid (AA, BB, DD), and although each of the genesis similar, partial differences are found due to translocation and thelike. On the other hand, durum wheat is tetraploid and does not containgenomic DD.

In terms of its genome structure and the base sequence of its encodedgenes, wheat shares a high degree of homology with other cereal grainssuch as barley, rye and oats. These grains have a high level of homologywith common wheat, and therefore the possibility of false detection ishigh.

Under these circumstances, it has been difficult in wheat to discover aDNA sequence satisfying the following four conditions: a) it isuniversally present in wheat varieties, b) the amount present (detectedamount) will not be affected depending on the wheat variety, c) even ifother grains are present, only wheat will be detected withoutcross-reactivity, and d) it will be amplified quantitatively by the PCRreaction.

WO 2005/097989 (Patent Document 1) discloses that a partial sequence ofthe Waxy gene (see Non-Patent Document 2), etc., can be used asendogenous wheat DNA satisfying such conditions.

-   Patent Document 1: WO 2005/097989-   Non-Patent Document 1: JAS Analytical Test Handbook, “Genetically    Modified Food Test and Analysis Manual for Individual Products,    Revised Second Edition”-   Non-Patent Document 2: Ainsworth C, et al., Plant Mol Biol. 1993    April; 22(1):67-82

DISCLOSURE OF INVENTION

However, waxy wheat, a mutant lacking the Waxy gene, is already known,and in test samples containing waxy wheat it is impossible to assay theendogenous wheat DNA accurately by detecting the partial sequence of theWaxy gene disclosed in Patent Document 1. In addition, since Waxy geneis thought to be present in the D genome of wheat, it will not bepresent in the genome of durum wheat, which lacks the D genome.Therefore, although the method disclosed in patent document 1 is usefulwhen the goal is to distinguish between durum wheat and common wheat,there are many cases requiring the detection of a total of durum wheatand common wheat as the detection of “wheat,” and under thosecircumstances an endogenous durum wheat DNA sequence must be discovered,detected and quantitated separately.

Therefore, an object of the present invention is to discover anadditional endogenous wheat sequence satisfying conditions a) to d)above, and a further object of the present invention is to provide amethod of accurately detecting or quantitating endogenous wheat DNA in atest sample by the polymerase chain reaction.

The inventors conducted diligent research to solve the above problem anddiscovered that a partial region of the proline rich protein (PRP) gene(C. A. Raines, et al., Plant Molecular Biology, 16: 663-670, 1991) inthe genomic DNA of wheat is universally present throughout wheatvarieties and does not cross-react with other plants in PCR reactions,and that it is possible to specifically detect and quantitate anendogenous wheat DNA sequence by amplifying that region, thus completedthe present invention.

More specifically, the present invention relates to:

(1) A method of detecting or quantitating endogenous wheat DNA in a testsample by a polymerase chain reaction, the method comprising: using anucleic acid in the sample or a nucleic acid extracted from the sampleas the template to amplify the nucleic acid of a region consisting ofthe base sequence identified as SEQ ID NO: 2 or a partial sequencethereof with a primer pair capable of amplifying that region; anddetecting or quantitating the amplified nucleic acid;(2) The method according to (1) above, wherein the region consisting ofthe partial sequence of the base sequence identified as SEQ ID NO: 2 isa region consisting of any one of the base sequences identified as SEQID NOs: 11 to 18;(3) The method according to (1) above, wherein the aforementioned primerpair is a primer pair selected from a group consisting of: (i) a primerpair consisting of a nucleic acid comprising the base sequenceidentified as SEQ ID NO: 3 and a nucleic acid comprising the basesequence identified as SEQ ID NO: 4, (ii) a primer pair consisting of anucleic acid comprising the base sequence identified as SEQ ID NO: and anucleic acid comprising the base sequence identified as SEQ ID NO: 4,(iii) a primer pair consisting of a nucleic acid comprising the basesequence identified as SEQ ID NO: 3 and a nucleic acid comprising thebase sequence identified as SEQ ID NO: 6, (iv) a primer pair consistingof a nucleic acid comprising the base sequence identified as SEQ ID NO:5 and a nucleic acid comprising the base sequence identified as SEQ IDNO: 6, (v) a primer pair consisting of a nucleic acid comprising thebase sequence identified as SEQ ID NO: 3 and a nucleic acid comprisingthe base sequence identified as SEQ ID NO: 7, (vi) a primer pairconsisting of a nucleic acid comprising the base sequence identified asSEQ ID NO: 3 and a nucleic acid comprising the base sequence identifiedas SEQ ID NO: 8, (vii) a primer pair consisting of a nucleic acidcomprising the base sequence identified as SEQ ID NO: 5 and a nucleicacid comprising the base sequence identified as SEQ ID NO: 8, (viii) aprimer pair consisting of a nucleic acid comprising the base sequenceidentified as SEQ ID NO: 5 and a nucleic acid comprising the basesequence identified as SEQ ID NO: 7, (ix) a primer pair consisting of apair of nucleic acids with each nucleic acid comprising a base sequencein common with at least 80% continuous base sequence of each nucleicacid in the primer pairs in (i) to (viii) above;(4) The method according to any one of (1) to (3) above, wherein eachprimer is the primer pair is a nucleic acid having 15 to 40 bases long;(5) The method according to any one of (1) to (4) above, wherein theregion amplified in the above polymerase chain reaction is detectedusing a probe identified as SEQ ID NO: 9 or 10;(6) A kit for detecting or quantitating an endogenous wheat DNA sequencein a test sample by the polymerase chain reaction, containing at leastone primer pair of the primer pairs according to (3) above;(7) Replicable DNA containing: a DNA sequence consisting of the basesequence identified as SEQ ID NO: 2 or a partial sequence thereof asendogenous DNA common to both genetically modified wheat andnon-genetically modified wheat, and one or more DNA sequences comprisingsequences that are specific to each strain of genetically modified wheatas genetically modified wheat-specific DNA;(8) Replicable DNA containing: a DNA sequence consisting of a basesequence having at least 80% homology with the DNA consisting of thebase sequence identified as SEQ ID NO: 2 or a partial sequence thereofas endogenous DNA common to both genetically modified wheat andnon-genetically modified wheat, and one or more DNA sequences consistingof sequences that are specific to each strain of genetically modifiedwheat as genetically modified wheat-specific DNA;(9) The method according to (7) or (8) above, wherein the DNA consistingof the partial sequence of the base sequence identified as SEQ ID NO: 2is DNA consisting of any one of the base sequences identified as SEQ IDNOs: 11 to 18;(10) Replicable DNA containing: DNA consisting of a sequence capable ofbeing amplified by the polymerase chain reaction using any of the primerpairs according to (3) above as endogenous DNA common to bothgenetically modified wheat and non-genetically modified wheat, and oneor more DNA sequences consisting of sequences that are specific to eachstrain of genetically modified wheat as genetically modifiedwheat-specific DNA;(11) A method of determining a contamination rate of geneticallymodified wheat in a test sample, the method comprising: a step ofpreparing two or more types of concentration dilution series ofsolutions containing replicable DNA according to any one of (7) to (10)above, performing quantitative polymerase chain reactions for each,amplifying the above endogenous wheat DNA and at least one type of theabove genetically modified wheat-specific DNA, and determining acalibration curve for each partial region; and a step of performing forthe test sample a quantitative polymerase chain reaction under the sameconditions as the above step for determining the calibration curves, anddetermining the number of the above endogenous wheat DNA molecules andthe number of the above genetically modified wheat-specific DNAmolecules present in the test sample using the above calibration curve;(12) The method according to (11) above further comprising a step ofdetermining the contamination rate of genetically modified wheat in thetest sample by calculating a formula 100×A/B using: the ratio A obtainedby dividing the number of the above genetically modified wheat-specificDNA molecules by the number of the endogenous wheat DNA moleculespresent in the above test sample, and the ratio B obtained by dividingthe number of DNA molecules specific to each strain of geneticallymodified wheat determined by performing quantitative polymerase chainreactions using standard seeds from each strain of genetically modifiedwheat by the number of endogenous wheat DNA molecules; and(13) The method according to (11) or (12) above wherein amplification ofthe above endogenous wheat DNA is performed using at least one primerpair selected from the primer pairs according to (3) above.

EFFECT OF THE INVENTION

In accordance with the present invention it is possible to specificallydetect and quantitate endogenous wheat DNA without cross reaction fromother crops in a test sample such as a food material, processed food,and the like by PCR amplification of a partial region of the PRP gene.

Furthermore, it is possible to determine with good precision thecontamination rate of each GMO strain in a test sample by quantitativePCR utilizing the standard reference material for detecting geneticallymodified wheat (hereinafter, sometimes referred to as “GM wheat”)provided by the method herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding regions in SEQ ID NO: 2 of the primersidentified as SEQ ID NOs: 3 to 8;

FIG. 2 shows the amplification curve and melting curves when real-timePCR is performed by the SYBR procedure using the standard plasmid pWIG03as a template on the amplified regions of the PRP and the Waxy genes;

FIG. 3 shows the results when real-time PCR was performed by the SYBRprocedure using DNA extracted from wheat seeds as the template and usingthe primer pairs of PRP01-F and PRP01R, PRP03-F and PRP03-R, and PRP03-Fand PRP07-R shown in Table 10;

FIG. 4 shows the detection efficiency by real-time PCR using thecombinations of primers shown in Table 11;

FIG. 5 shows the detection Examples in 20 of 40 varieties of wheatwherein PRP03 was detected;

FIG. 6 shows the results when the hypothetical GM wheat contaminationrate was determined using the results obtained when DNA was extractedfrom a sample wherein the hypothetical GM wheat was arbitrarily mixedwith separately pulverized wheat (Yecora Rojo variety), and using thisDNA as a template and following the method of the present invention, theendogenous wheat gene and Roundup® resistance gene were quantitated; and

FIG. 7 shows the results of the verification test for cross-reactivitywith other crops performed using two types of primer pairs, PRP03-F andPRP03-R, and PRP03-F and PRP07-R, and PRP-Taq2 as the probe.

DETAILED DESCRIPTION

Below the meanings of terms used in this description are defined, andthe present invention is described in detail.

In this description the term “wheat” refers to common wheat, durumwheat, and waxy wheat unless specifically stated otherwise.

The method of the present invention detects a specific part of the PRPgene (GeneBank Accession No. X52472, SEQ ID NO: 1) in the wheat genome,and more specifically, the region consisting of the base sequenceidentified as SEQ ID NO: 2 or a partial sequence thereof, as theendogenous wheat DNA sequence.

When the Wx012 region disclosed in Patent Document 1 and a plurality ofpartial regions of the PRP gene were amplified by PCR, it was found thatin the various varieties of wheat the ratio of the Wx012 regionamplification product to the amplification products of the variousregions of the PRP gene varies between 1:1 and 1:3. Because it isthought that the ratios other than 1:1 are due to the presence of morethan two regions on the PRP gene that are amplified by the same primer,these regions are not suitable for use as the endogenous wheat sequenceused in the present invention. Therefore, the inventors continued theirinvestigation and found that if a region consisting of the base sequenceidentified as SEQ ID NO: 2 or a partial sequence thereof (providing itis a region of 80 bases or more) is amplified, that amplificationproduct and the Wx012 amplification product are obtained in a 1:1 ratio.

In addition, it was verified that the base sequence identified as SEQ IDNO: 2 or partial sequence thereof is detected in durum wheat as well.Therefore, it can be assumed that the PRP gene is present on wheatgenome A or B. Since the wheat currently distributed on the marketincludes common wheat, durum wheat, waxy wheat, etc., as the endogenouswheat gene to be detected, it is preferred that the gene be universallypresent in these varieties of wheat and the amount present does not varydepending on the variety. In this regard, because genomes A and B mustboth be present in these varieties of wheat, the region consisting ofthe base sequence identified as SEQ ID NO: 2 is preferred as anendogenous wheat gene.

On the other hand, it was confirmed that a pseudogene that is extremelysimilar to the PRP gene exists. Therefore, in the process of amplifyingthe wheat genome in a processed food, if a primer that amplifies apartial sequence selected randomly from the entire length of the PRPgene is used, it is possible that the partial sequence of the pseudogenewill also be amplified simultaneously. In other words, it is assumedthat depending on the variety of wheat, there will be cases in which thequantity of amplified DNA per unit of wheat will inconsistent. However,the inventors have verified that if PCR is performed using a primer thatamplifies the region consisting of the base sequence identified as SEQID NO: 2 or a partial sequence thereof, only that region of the PRP genewill be amplified with no pseudogene cross-reactivity.

Additionally, the region consisting of the base sequence identified asSEQ ID NO: 2 is an extremely short 330 bp compared with the total genomelength, and therefore it is possible to detect and quantitate endogenouswheat DNA from a sample of a processed food, etc., in which the DNA maybe fragmented.

The partial sequence of the base sequence identified as SEQ ID NO: 2 isnot particularly limited in the present invention, but preferably thelength of the sequence will be at least 80 bases, and any one of thebase sequences identified as SEQ ID NOs: 11 to 18 is preferred. Thesesequences can be amplified with PCR by suitably combining primersconsisting of nucleic acids comprising the base sequences identified asSEQ ID NOs: 3 to 8.

FIG. 1 shows the binding regions of the primers identified as SEQ IDNOs: 3 to 8 in SEQ ID NO: 2.

By altering the base length of the primers identified as SEQ ID NOs: 3to 8 or by slightly altering the primer binding region, the amplifiedsequence becomes a region slightly displaced from the region comprisingthe base sequences identified in SEQ ID NOs: 11 to 18, but those regionsare included within the “partial sequence of the base sequenceidentified as SEQ ID NO: 2” in the present invention. An example of sucha region is a region having at least 80% or more, and preferably 90% ormore, in common with the base sequences identified in SEQ ID NOs: 3 to8.

The primer pair used in the PCR method in the present invention is notparticularly limited provided it is a primer pair that can amplify aregion consisting of the base sequence identified as SEQ ID NO: 2 or apartial sequence thereof, and the primer pair can be designed accordingto the basic rules for primer preparation in accordance with the basesequence of the region to be amplified. During that process unificationof the Tm values of the primers should be kept in mind. The length ofeach primer should normally be 15 to 40 bp, and preferably 15 to 30 bp.

In the method of the present invention, if the primer pair to be usedcross-reacts with a crop other than wheat, not only will the possibilityof a false positive detection of wheat arise, but also it will beimpossible to accurately quantitate the endogenous wheat DNA sequence inthe test sample. The method of the present invention provides accurateinformation on the presence of wheat in a test sample such as a foodmaterial, processed food, etc., and the quantity thereof. Therefore, theprimer pair for PCR used in the method of the present invention must notcross-react with other crops such as rice, barley, rye, oats, rapeseed,maize, foxtail millet, kibi millet, buckwheat, etc.

Examples of such primer pairs include (i) a primer pair consisting of anucleic acid comprising the base sequence identified as SEQ ID NO: 3 anda nucleic acid comprising the base sequence identified as SEQ ID NO: 4,(ii) a primer pair consisting of a nucleic acid comprising the basesequence identified as SEQ ID NO: 5 and a nucleic acid comprising thebase sequence identified as SEQ ID NO: 4, (iii) a primer pair consistingof a nucleic acid comprising the base sequence identified as SEQ ID NO:3 and a nucleic acid comprising the base sequence identified as SEQ IDNO: 6, (iv) a primer pair consisting of a nucleic acid comprising thebase sequence identified as SEQ ID NO: 5 and a nucleic acid comprisingthe base sequence identified as SEQ ID NO: 6, (v) a primer pairconsisting of a nucleic acid comprising the base sequence identified asSEQ ID NO: 3 and a nucleic acid comprising the base sequence identifiedas SEQ ID NO: 7, (vi) a primer pair consisting of a nucleic acidcomprising the base sequence identified as SEQ ID NO: 3 and a nucleicacid comprising the base sequence identified as SEQ ID NO: 8, (vii) aprimer pair consisting of a nucleic acid comprising the base sequenceidentified as SEQ ID NO: 5 and a nucleic acid comprising the basesequence identified as SEQ ID NO: 8, (viii) a primer pair consisting ofa nucleic acid comprising the base sequence identified as SEQ ID NO: 5and a nucleic acid comprising the base sequence identified as SEQ ID NO:7, (ix) a primer pair consisting of a pair of nucleic acids with eachnucleic acid comprising a continuous base sequence having at least 80%or more, preferably 90% or more, and more preferably 85%, in common withthe base sequence of a nucleic acid in each primer pair in (i) to (viii)above. These primer pairs can specifically amplify a region comprisingthe base sequence identified as SEQ ID NO: 2 or a partial sequencethereof without cross-reacting with crops other than wheat.

In the method of the present invention the probe for detecting theregion to be amplified by PCR is not particularly limited provided itcan detect the amplification product quantitatively, but it ispreferable to perform detection using a probe comprising the basesequence identified as SEQ ID NO: 9 or 10. These probes bind with highspecificity to the amplification product, i.e., part of the regionconsisting of any of the base sequences identified as SEQ ID NOs: 11 to18, and therefore they can detect the amplification productquantitatively. FIG. 1 shows the region to which a probe consisting ofthe base sequence identified as SEQ ID NO: 9 or 10 will bind.

The test sample used in the present invention is a food material orprocessed food that contains or may contain wheat, and examples includefood materials and processed intermediate materials thereof such as rawgrains of wheat, dried grains, wheat flour, mixed flour, and the like,and processed foods such as bread, noodles, and the like. The foodmaterial or food product includes not only human food products but alsopet food and animal feed. Additionally, the term crops other than wheatrefers to all crops that can be used as a food material or food rawmaterial and include, for example, the aforementioned crops.

A test sample may be subjected to extraction of nucleic acid as is orafter pulverized, or may be subjected to extraction of nucleic acidafter washed, dried, and then pulverized. The nucleic acid extractedfrom the test sample used in analysis is normally DNA. The DNA may beextracted by a publicly known desired method. At present many DNAextraction kits are on the market, and extraction can be performed usingsuch a kit. For example, DNA can be extracted from a test sample usingthe DNeasy Plant Maxi Kit (QIAGEN GmbH) following the method of Koppel,et al., (Kopell, E. et al., Mitt. Gebiete Levensm, Hyg., 88, 164). Theconcentration of extracted DNA can be determined by an absorbancemeasurement, etc., and preferably is diluted to a concentration optimalfor PCR and used.

In the method of the present invention PCR can be performed according tothe conventional manner in consideration of the primers and DNApolymerase to be used. During that process, the PCR buffer solution,dNTP, reagents such as MgCl₂ and the like can be prepared, or acommercially available PCR kit can be used. One or two or more of theabove primer pairs can be used in the PCR. An example of PCR conditionsis 40 cycles with one cycle being 30 sec at 95° C., 30 sec at 63° C.,and 30 sec at 72° C., with the final reaction being 7 min at 72° C.However, these conditions can be suitably varied in consideration of theTm of the primers to be used, the length of the region to be amplified,concentration of template DNA, and the like.

Detection of the amplified nucleic acid (PCR product) can be performedusing a desired method that can identify a specific DNA fragment, morespecifically, methods including agarose gel electrophoresis, acrylamidegel electrophoresis, capillary electrophoresis, hybridization,immunological methods, and the like. Generally, the result is verifiedby the migration pattern of the electrophoresis performed with the PCRproduct. For example, electrophoresis on a 0.8% agarose gel containingethidium bromide can be performed and detection can be accomplished byband verification.

The present invention includes the primer pairs used in theaforementioned detection and quantitation method, and a kit containingthose primer pairs. The primers can be produced by conventional methods.In addition to the primer pairs, the kit can contain other reagents,e.g., dNTP, MgCl₂, a polymerase such as TaqDNA polymerase, buffersolution (for example, Tris-HCl), glycerol, DMSO, DNA for a positivecontrol, DNA for a negative control, distilled water, and the like. Inthe kit these reagents may be provided in individual packages, or two ormore types of reagents may be provided as a mixture thereof. Theconcentrations of each of the reagents in the kit are not particularlylimited in the present invention provided they are within a permissiblerange for performance of the PCR method in the present invention.Information on optimal PCR conditions and the like may also be includedin the kit, or the kit may contain only the primer reagents.

The present invention provides a standard reference material useful whenmeasuring the GM wheat contamination rate by quantitative PCR. Thisstandard reference material is made by ligating endogenous DNA common toboth non-GM wheat and GM wheat and DNA specific to at least one GM wheaton a single replicable DNA molecule. DNA comprising a base sequenceidentified as any one of SEQ ID NOs: 11 to 18 can be used as theaforementioned partial sequence.

The standard reference material of the present invention may also be areplicable DNA molecule comprising DNA consisting of a base sequencehaving at least 80% homology with DNA consisting of the base sequenceidentified as SEQ ID NO: 2 or a partial sequence thereof as endogenousDNA.

The replicable DNA used as the standard reference material is notparticularly limited in the present invention provided endogenous DNAand DNA specific to a strain of GM wheat can be inserted thereinto, anda commercially available vector such as a pBR-series vector (e.g.,pBR322, pBR328, etc.), a pUC-series vector (pUC19, pUC18, etc.), a λphase series vector (λgt10, λgt11, etc.), or a modified form thereof canbe used.

In the case of detecting GM wheat, it is not only necessary to amplifyand detect a foreign DNA sequence inserted into the normal wheat genomeby genetic engineering, but also to amplify a region containing theendogenous sequences upstream and downstream of the foreign DNA. BecauseGMO are prepared in other crops as well by inserting the same foreignDNA sequence, if the foreign DNA sequence alone is detected, it is stillimpossible to judge whether it originates in GM wheat or in anothergenetically modified crop. Therefore, primers for detecting a sequencespecific to a GMO strain must be primers that can amplify the regioncontaining the foreign DNA sequence inserted into each strain of GMwheat, and also the upstream and downstream endogenous sequences. Thesekinds of primers are prepared, for example, by following theaforementioned method of Koppel et al., reported for soybeans, themethod of Wurz et al., (Wurz, A. et al.; 2nd Status report. BgVV,BgVV-Heft, 1/199797,118), or a method based thereon. The sequencespecific to each strain of GM wheat inserted in the aforementionedstandard reference material is selected from DNA sequences that can beamplified by these primers.

After the endogenous wheat DNA and the GM wheat-specific DNA to beinserted into the standard reference material are determined, PCR isperformed using the normal wheat genome or GM wheat genome as atemplate, the endogenous DNA and the GM wheat-specific DNA are cloned,and by cleaving the cloned DNA fragments and the cloning site of theaforementioned replicable DNA with the same restriction enzyme, the DNAfragments can be ligated into the cleavage site in the replicable DNA.Publicly known restriction enzymes can be suitably selected and used,and examples include EcoRI, SpeI, EcoRV, SmaI, SacI, NotI, HindIII,XhoI, etc.

If two or more types of dilution series of the above solution containingthe standard reference material are prepared and quantitative PCR isperformed on each, it is possible to determine calibration curves forthe partial regions of both the endogenous wheat DNA sequence and theGMO-specific DNA sequence. The standard reference material of thepresent invention can also be used as a positive control for theendogenous wheat DNA sequence and GMO-specific DNA sequence inqualitative PCR.

The present invention includes a method of determining the GM-wheatcontamination rate in a test sample by PCR using the above standardreference material. This method comprises the step of determining thecalibration curve of a specific sequence using the above standardreference material, and the step of performing a quantitative polymerasechain reaction under the same conditions used when determining thecalibration curve, amplifying the partial region of the endogenous wheatDNA sequence and the partial region of the GM wheat-specific DNAsequence, and by using the calibration curves, determining the number ofmolecules of the partial region of the endogenous wheat DNA sequence andof the partial region of the GM wheat-specific DNA sequence.

When calculating the contamination rate of GM wheat in a test sample,first ratio A is determined: Ratio A is obtained by dividing the numberof molecules of the partial region of the GM wheat-specific DNA sequenceby the number of molecules of the partial region of the endogenous wheatDNA sequence. Then ratio B is determined: Ratio B is obtained bydividing the number of molecules of the partial region of the DNAsequence specific to each strain of GM wheat, which is obtained byperforming quantitative PCR using a standard grain of geneticallymodified wheat, by the number of molecules of the partial region of theendogenous wheat DNA sequence. Finally, it is possible to perform thestep of determining the contamination rate of genetically modified wheatin the test sample by calculating the formula 100×A/B. The above ratio Bis called the “internal standard ratio” in Non-Patent Document 1, and itis the ratio signifying (recombinant gene)/(endogenous gene) in the DNAextracted from the grains of each pure GM strain.

Each PCR step performed in the method of determining the GM wheatcontamination rate in the present invention can be performed separately,but they may be performed simultaneously. It is preferred that each PCRstep be performed under conditions wherein the nucleic acidamplification reaction occurs at approximately the same rate as in thePCR performed to prepare the calibration curves. Examples of suchconditions are ones wherein the temperatures and cycles are the same asin the PCR performed to prepare the calibration curves. The calculationof the GM wheat contamination rate may be performed by measuring theamounts of endogenous DNA and recombinant DNA separately and calculatingthose measured results, but it is also possible to perform amplificationwith a real-time PCR apparatus following the method described inNon-Patent Document 1 and calculate the GM wheat contamination ratethereby. In the present invention the term “recombinant DNA” refers toan arbitrary foreign DNA molecule artificially inserted into wheat, andexamples include DNA of a region encoding a foreign gene, anuntranscribed or untranslated region, linker region, or vector part.

EXAMPLES

The present invention is explained in greater detail through thefollowing examples, but the present invention is by no means limitedthereto, and a variety of embodiments thereof are possible. Therefore,it should be understood that the present invention encompasses all suchembodiments thereof provided they are in accordance with the conceptsdisclosed by the description and drawings herein.

The following samples, reagents, and devices are used in examples below.

(1) Samples

Wheat (Triticum aestivum): dried seeds of the following varieties wereused: Hank, Scarlet, Tara, Yecora Rojo, Finch, Lewjain, Rod,Weatherford, Estica, Buchanan, Finley, Garland, TAM 107, Declo, Hatton,Morgan, Neely, Ramp, Amidon, Ernest, McNeal, AC-Barrie, AC-Splendor,CDC-Teal, Laura, Nishikaze, Nishihonami, Chikugo Izumi, Tsugaru HDC,Shirogane, Shirasagi, Bihoro HDN, Hokushin, Bandowase, Kita HDI, NorinNo. 61, Hatsumochi, Mochiotome, Kanto 107, and Bai Huo.

Durum wheat (Triticum durum): dried seeds of the following variety wereused: AC Navigator.

Maize (Zea mays): dried seeds of the following variety were used: Dentcorn.

Soybeans (Glycine max): dried seeds of the following variety were used:descendent variety of genetically modified soybean Roundup® Ready Soy.

Rice (Oryza sativa): dried seeds of the following variety were used:Koshihikari.

Barley (Hordeum vulgare): dried seeds of the following variety wereused: Benkei.

Oats (Avena sativa): dried seeds of the following variety were used:Commercial product.

Rye (Secale cereale): dried seeds of the following variety were used:Commercial product.

Rapeseed (Brassica napus): dried seeds of the following variety wereused: Canola.

Foxtail millet (Setaria italica Beauvois): dried seeds of the followingvariety were used: Mochiawa.

Kibi millet (Panicum miliaceum Panicum): dried seeds of the followingvariety were used: Mochikibi.

Sorghum (Sorghum subglabrescens): dried seeds of the following varietywere used: Commercial product.

Buckwheat (Fagopyrum esculentum): dried seeds of the following varietywere used: Domestic variety.

(2) Reagents

(2-1) The following reagents were used for the extraction of DNA fromthe samples.

Sodium lauryl sulfate (SDS) (special reagent grade) (Sigma Chemical Co.)

QIAGEN DNeasy Plant Maxi Kit (QIAGEN GmbH)

QIAGEN DNeasy Plant Mini Kit (QIAGEN GmbH)

The following reagents were used for DNA electrophoresis.

Acetic acid (special reagent grade) (Wako Pure Chemical Industries,Ltd.)

Tris (hydroxymethyl) aminomethane (Tris) (special reagent grade) (SigmaChemical Co.)

Ethylenediaminetetraacetate (EDTA) (special reagent grade) (SigmaChemical Co.)

Agarose powder “TaKaRa LO3” (TaKaRa Shuzo Co., Ltd.)

Ethidium bromide (Sigma Chemical Co.)

Bromophenol blue (Sigma Chemical Co.)

Xylene cyanol (Sigma Chemical Co.)

DNA marker “1 kb ladder” (New England Biolabs Inc.)

DNA marker “100 bp ladder” (New England Biolabs Inc.)

(2-2) The following reagents were used for qualitative PCR.

DNA polymerase “AmpliTaq Gold” (Applied Biosystems)

×10 PCR Buffer II (Applied Biosystems)

(2-3) The following reagents were used for plasmid preparation andpurification.

DNA polymerase “AmpliTaq Gold” (Applied Biosystems)

×10 PCR Buffer II (Applied Biosystems)

DNA polymerase “KOD” (TOYOBO Co., Ltd.)

×10 PCR Buffer II (TOYOBO Co., Ltd.)

TOPO TA Cloning Kit with TOP10F′ Cells (Invitrogen Co.)

Yeast extract (Difco Laboratories)

Tryptone Peptone (Difco Laboratories)

NaCl (special reagent grade) (Wako Pure Chemical Industries, Ltd.)

Agarose powder (TAKARA BIO, Inc.)

D[−]-α-Aminobenzylpenicillin (Ampicillin) Sodium Salt (Sigma ChemicalCo.)

QIAGEN Plasmid Maxi Kit (QIAGEN GmbH)

Ethanol (special reagent grade) (Wako Pure Chemical Industries, Ltd.)

2-Propanol (special reagent grade) (Wako Pure Chemical Industries, Ltd.)

Tris (hydroxymethyl) aminomethane (Tris) (special reagent grade) (SigmaChemical Co.)

Ethylenediaminetetraacetate (EDTA) (special reagent grade) (SigmaChemical Co.)

Restriction enzyme “EcoRI” (TaKaRa Shuzo CO., LTD.)

Restriction enzyme “SacI” (New England Biolabs Inc.)

Restriction enzyme “XbaI” (New England Biolabs Inc.)

Calf Intestinal Alkaline Phosphatase (Invitrogen)

Phenol (special reagent grade) (Wako Pure Chemical Industries, Ltd.)

Chloroform (special reagent grade) (Wako Pure Chemical Industries, Ltd.)

Isoamyl alcohol (special reagent grade) (Wako Pure Chemical Industries,Ltd.)

(2-4) The following reagent was used for quantitative PCR.

TaqMan Universal PCR Master Mix (Applied Biosystems)

(3) Equipment

(3-1) The following equipment was used for the extraction of DNA fromthe samples.

Pulverizer “Multi Beads Shocker MB301” (Yasui Kikai Co., Ltd.)

(3-2) The following equipment was used for DNA electrophoresis.

Electrophoresis device “Mupid 2” (Advance Co., Ltd.)

(3-3) The following equipment was used for qualitative PCR.

Thermal cycler “PTC-200” (MJ Research Inc.)

(3-4) The following equipment was used for quantitative PCR.

Quantitative PCR device “ABI PRISM 7700 Sequence Detector System”(Applied Biosystems)

Synthesis of the primers and probes was consigned to OperonBiotechnologies.

Example 1 Plasmid Construction

Genomic DNA was extracted from grains of the Ernest cultivar of wheat,and PCR was performed using this DNA as a template. Table 1 shows thetargets of amplification, and amplification was performed for two typesof endogenous wheat gene candidates (PRP gene and Waxy gene), and forthe RRS gene as a hypothetical GM wheat gene. Table 2 shows the primersthat were used. As explained below, the RRS gene is a Roundup® resistantgene.

TABLE 1 Gene General Name Size (bp) PRP Proline rich protein 259 WaxyWaxyD1 529 RRS Enolpymylshikimate phosphate synthase 1886

TABLE 2 Amplicon Gene Name Sequence size PRP prp04-F5′-GCCTCCGAAGGGCAAGC-3′  259 bp (SEQ ID NO: 5) prp06-R5′-GAACATATACCAACACGCAAATG-3′ (SEQ ID NO: 6) RRS RRS-F5′-TGGAAAAGGAAGGTGGCTCCTAC-3′ 1919 bp (SEQ ID NO: 23) RRS-R5′-GGGAATTGGATCCGGTACCGA-3′ (SEQ ID NO: 24) RRS Sac-F5′-AGAGCTCTGGAAAAGGAAGGTGGCTCCTAC-3′ 1926 bp (SEQ ID NO: 25) RRS Sac-R5′-CGGAATTCGATCCGGTACCGA-3′ (SEQ ID NO: 26) Waxy Wx-F5′-TTTTGTTGTGCCGCTTGCCT-3′  529 bp (SEQ ID NO: 27) Wx-R5′-AGTTTAGCGCGTCACAGACTCA-3′ (SEQ ID NO: 28) Wx Xba-F5′-TCTCTAGATTTTGTTGTGCCGCTTGCCT-3′  545 bp (SEQ ID NO: 29) Wx Wba-R5′-TCTCTAGAGTTTAGCGCGTCACAGACTCA-3′ (SEQ ID NO: 30)

Next, PCR was performed again using the obtained amplified DNA as atemplate with a primer having restriction enzyme cleavage site addedthereto. After digestion with the restriction enzyme had been performedon the obtained amplified DNA, the purified fragments were ligated topUC19 vectors, and E. coli were transformed thereby. Alternatively, TAcloning into a pCR4 vector was performed. After the insertion fragmentswere verified by restriction enzyme mapping, the entire sequences of theobtained clones were verified by sequencing.

After restriction enzyme digestion of each of the TA clones, thepurified fragments were ligated to pUC19 vectors, and E. coli weretransformed thereby.

The plasmids were extracted from the transformants and purified, and thesequences were verified.

Plasmids were constructed according to the process described above. Morespecifically, the WaxyD1 region amplified with a primer having arestriction enzyme cleavage site added thereto was digested with XbaI,and then it was ligated to pUC19 that had been subjected to XbaIdigestion in the same manner and dephosphorylated to construct pWIG01.

TA cloning was performed once on the PCR-amplified RRS gene, theobtained plasmid pRRS was digested with EcoRI and SacI, and the RRSfragment was purified. In the same manner, pWIG01 was digested withEcoRI and SacI, and after the plasmid was purified, the RRS fragment wasligated thereto to construct pWIG02.

TA cloning was performed once on the PCR-amplified PRP gene, theobtained plasmid pPRP was digested with EcoRI, and the PRP fragment waspurified. In the same manner, pWIG02 was digested with EcoRI, anddephosphorylated, and then the PRP fragment was ligated thereto toconstruct pWIG03.

Sequencing of the constructed plasmids was performed and the insertionsequences were verified. All verified sequences matched the targetsequences.

The procedure used in Examples 1 to 3 is specifically described below.

(1) Restriction Enzyme Digestion

The procedures were performed following the manuals for each restrictionenzyme. More specifically, each DNA solution was mixed with therestriction enzyme, distilled water, and the ×10 buffer for the enzyme,and the reaction was normally carried out for 2 h at 37° C.

(2) DNA Fragment Purification Following Digestion

The DNA fragments were separated using agarose gel electrophoresis. Thekit from QIAGEN was used for purification from the gel. In other words,the gel containing the target DNA was heated and dissolved, and the DNAwas bound to a silica film. After the silica film was rinsed with asolution containing ethanol, etc., the DNA was eluted with distilledwater.

(3) Dephosphorylation of Restriction Enzyme-Digested Plasmids

The plasmids were subjected to restriction enzyme digestion anddesalted. Then the plasmids were mixed with calf intestinal alkalinephosphatase (CIAP, GIBCO BRL) and a dedicated buffer, and reacted for 30min at 37° C. The CIAP was deactivated after the reaction by treatmentwith phenol, and the dephosphorylated plasmid was recovered by ethanolprecipitation.

(4) DNA Ligation

A DNA ligation kit (TAKARA BIO INC.) ver. 2 was used. More specifically,the target DNA was mixed into solution, an equivalent volume of the kitreaction solution (solution I) was added, and the mixture was let standfor 30 min at 16° C. to perform DNA ligation.

(5) Transformation

E. coli DH5a competent cells (Toyobo Co., Ltd.) were used. A thawedsuspension of 10 to 50 μL of competent cells was mixed with DNA and letstand on ice for 30 min, and after a heat shock for 50 sec at 42° C.,the mixture was placed on ice again, 450 μL of SOC medium warmed to 37°C. was added after 2 min had elapsed, and the mixture was incubated for1 h at 37° C. The incubate was then spread at 100 μL/plate onCirclegrow® Amp+ medium plates and cultured for 16 h at 37° C.

(6) Culturing

Culturing for the purpose of plasmid purification was performed usingCirclegrow® medium. Ampicillin resistance was used for the plasmidproduction selection pressure, and a final concentration of 100 μg/mL ofampicillin was used. Culturing was performed for 14 to 16 h at 37° C.using a test tube shaker.

(7) PCR

AmpliTaq Gold polymerase from Applied Biosystems was used. Thecomposition shown in Table 3 was used as the reaction composition. Thereaction was performed under the conditions shown in Table 4.

TABLE 3 μL 10 × Buffer 2.5 25 mM MgCl₂ 1.5 2.5 mM dNTP 2.0 Primer pair2.5 5 u/mL Taq 0.25 MQ H₂O 15.75 50 ng/μL DNA 0.5 Total 25.0

TABLE 4 Step Temp. (° C.) Time (min) Cycle No. 1 95 10 min 2 95 30 sec40 3 60 30 sec 4 72  2 min 5 72  7 min 6 10 ad infinitum

(8) TA Cloning

TA cloning was performed using the TOPO TA cloning system fromInvitrogen according to the manufacturer's manual.

(9) DNA Sequencing

DNA sequencing was performed using a model CEQ8000 from Beckman Coulteraccording to the manufacturer's manual. DTCS Quick Start Master Mix fromInvitrogen was used as the kit.

(10) Real-Time PCR SYBR Procedure

Real-time PCR was performed using a kit from TAKARA BIO (TAKARA SYBR®Premix Ex Taq™ (Perfect Real Time) Code No.: RR041A).

1) The template DNA (plasmid) was diluted in accordance with Table 5 (A5 ng/μL solution of ColE1 was used for the dilution).

2) Unknown DNA was prepared at 4 points in a dilution series rangingfrom 10-fold to 2-fold (A 5 ng/μL solution of ColE1 was used for thedilution).

3) The Master Mix was prepared for each primer according to Table 5.

4) The Master Mix and template DNA were mixed.

5) The reaction was initiated under the conditions shown in Table 6.

TABLE 5 Step Temp. Time Cycle Ramp time 1 50° C.  2 min 1 Auto 2 95° C.10 min 1 Auto 3 95° C.  5 sec 40 Auto 60° C. 30 sec Auto 4 95° C. 15 sec1 Auto 5 60° C. 20 sec 1 Auto 6 95° C. 15 sec 1 20 min 7 20° C.  1 min 1Auto

TABLE 6 Conc. μL SYBR Premix 5 Primer pair 5 μM each 0.4 ROX x50 0.2 MQH₂O 0.4 template 4.0 Total 10.0

(11) Real-Time PCR TaqMan° Procedure

Real-time PCR was performed using a kit from TAKARA BIO (TAKARA PremixEx Taq™ (Perfect Real Time) Code No.: RR039A).

1) The genomic DNA of each variety was used as the template withoutdilution.

2) The Master Mix was prepared by mixing with Premix, ROX, primer, andprobe according to Table 7.

3) The Master Mix at 16 μL/well was mixed with template DNA at 4μL/well.

4) The reaction was initiated under the conditions shown in Table 8.

TABLE 7 Conc. μL Premix  x2 10 ROX x50 0.4 Probe 10M 0.8 Primer pair  5Meach 0.8 H₂O 4.0 Total 16.0

TABLE 8 Step Temp. Time Cycle Ramp time 1 50° C.  2 min 1 Auto 2 95° C.10 min 1 Auto 3 95° C.  5 sec 40 Auto 60° C. 30 sec Auto 4 20° C.  1 min1 Auto

The template DNA (plasmid) was diluted in accordance with Table 9 (A 5ng/μL solution of ColE1 was used for the dilution).

TABLE 9

(12) Pulverization of Seeds (Small Quantity)

The aforementioned pulverizer, a Multi Beads Shocker MB301, was used.

1) One seed was placed in a 2 mL tube, a metal cone was added, and thetube was capped.

2) Pulverization was performed twice at 2000 rpm for 10 sec.

3) DNA was directly extracted from the pulverized powder.

(13) Pulverization of Seeds (Large Quantity)

A mill was used for pulverization.

1) Seeds (30 g) were placed in the mill and the lid was attached.

2) Pulverization was performed twice for 30 sec.

3) The pulverized powder was stored.

(14) Preparation of Genomic DNA

A kit from QIAGEN (QIAGEN Plant Mini Kit) was used. The procedure wasperformed according to the kit manual.

1) 400 μL of AP1 solution and 4 μL of RNase A were added to thepulverized seed, and the mixture was agitated.

2) The mixture was incubated for 10 min at 65° C., and mixed 2 or 3times during incubation.

3) 130 μL of AP2 was added, and after agitation, the mixture was letstand on ice for 10 min.

4) Centrifugal separation (15,000 rpm=20,000 g, 5 min, room temperature)was performed.

5) The entire volume of supernatant was placed in a QIAshredder, andcentrifugal separation (15,000 rpm, 2 min, room temperature) wasperformed.

6) The pass-through supernatant from decanting was transferred to aseparate container, 1.5 volumes (675 μL) of AP3/E was added, and themixture was agitated.

7) Half the volume was applied to a spin column, and centrifugalseparation (10,000 rpm, 1 min, room temperature) was performed.

8) The flow-through was discarded, and the same treatment was performedon the residual amount.

9) The column was placed in a new tube, 500 μL of AW was added, andcentrifugal separation (10,000 rpm, 1 min, room temperature) wasperformed.

10) After retreatment, the flow-through was discarded, and centrifugalseparation (15,000 rpm, 2 min, room temperature) was performed.

The column was placed in a fresh 1.5 mL tube, 50 μL of AE was added, andafter the mixture was let stand for 5 min at room temperature,centrifugal separation (10,000 rpm, 1 min, room temperature) wasperformed.

Thereafter, the above step was repeated.

(15) DNA Quantitation

A GeneSpec was used as the instrument, and DNA quantitation wasperformed according to the instrument manual.

The cell was 5 mm, the dilution rate was 1-fold, and the control was kitelution solution (AE).

Example 2 Verification of PCR Amplification Rate

For the amplification regions of the PRP and Waxy genes, real-time PCRwas performed by the SYBR procedure using the standard plasmid pWIG03 asa template. Template concentration-dependent amplification was observedfor all genes and all PCR regions under the same conditions. Accordingto the melting curves of the amplification products, each amplificationproduct had a main peak having a high-melting point, and no formation ofprimer dimers was observed. FIG. 2 shows the amplification curve andmelting curves. Table 10 shows a listing of the primer sequences used inExample 2 and thereafter.

TABLE 10 Amplicon size Gene Name Sequence (bp) PRP prp03-F5′-aag cca ccg atg act gac aat-3′ 231 (SEQ ID NO: 3) (SEQ ID NO: 11)prp07-R 5′-cgc aaa tga taa tta cag aat agt agt ac-3′ (SEQ ID NO: 4)prp04-F 5′-gcc tcc gaa ggg caa gc-3′ 244 (SEQ ID NO: 5) (SEQ ID NO: 12)prp07-R 5′-cgc aaa tga taa tta cag aat agt agt ac-3′ (SEQ ID NO: 4)prp03-F 5′-aag cca ccg atg act gac aat-3′ 246 (SEQ ID NO: 3)(SEQ ID NO: 13) prp06-R 5-gaa cat ata cca aca cgc aaa tg-3′(SEQ ID NO: 6) prp04-F 5′-gcc tcc gaa ggg caa gc-3′ (SEQ ID NO: 14)(SEQ ID NO: 5) prp06-R 5-gaa cat ata cca aca cgc aaa tg-3′(SEQ ID NO: 6) prp03-F 5′-aag cca ccg atg act gac aat-3′ 121(SEQ ID NO: 3) (SEQ ID NO: 15) prp03-R 5′-cgc taa ccg gat act atg cca-3′(SEQ ID NO: 7) prp03-F 5′-aag cca ccg atg act gac aat-3′  88(SEQ ID NO: 3) (SEQ ID NO: 16) prp04-R 5′-ata gca cat cgt ggc tcc gg-3′(SEQ ID NO: 7) prp04-F 5′-gcc tcc gaa ggg caa gc-3′ 101 (SEQ ID NO: 5)(SEQ ID NO: 17) prp04-R 5′-ata gca cat cgt ggc tcc gg-3′ (SEQ ID NO: 8)prp01-F 5′-gcg aca ccc cat cca ctt ta-3′ (SEQ ID NO: 19) prp01-R5′-cac ggc aag gag gct gtg-3′ (SEQ ID NO: 20) Waxy Wx012-F5′-ggt cgc agg aac aga ggt gt-3′ 102 (SEQ ID NO: 21) (SEQ ID NO: 18)Wx012-R 5′-ggt gtt cct cca ttg cga aa-3′ (SEQ ID NO: 22)

Example 3 Search for Amplified Regions in PRP Gene

A search for regions in the PRP gene of which the detected quantity doesnot vary depending on the variety of wheat was made. The Waxy gene wasused as a positive control, and a search for regions that give thedetective amount equivalent of Wx012 amplified regions was made. Theprimer pairs used were: PRP01-F and PRP01-R, PRP03-F and PRP03-R, andPRP03-F and PRP07-R shown in Table 10. Real-time PCR was performed bythe SYBR procedure using DNA extracted from wheat seeds as a template.

The results are shown in FIG. 3. When the primer pairs of PRP03-F andPRP03-R, and PRP03-F and PRP07-R were used, it was confirmed that thedetected amount of PRP was approximately the same as that of Wx012.However, when PRP01-F and PRP01-R were used as primer, the detectedamount was approximately three times larger than Wx012.

From this finding, it was confirmed that a gene homologous to PRP geneis present at the upstream region of the PRP gene (the amino-terminusside as a post-translation protein sequence) in wheat DNA, and thisupstream region is undesirable as endogenous DNA.

Example 4 Probe Search

From the investigations in Examples 1 to 3 it was confirmed that theregion from PRP04-F to PRP06-R is preferred as a region of an endogenouswheat gene for a PCR amplification. Probes were then designed that weresuitable to each type of primer pair for this region. For design of theprobes the Primer Express or Primer3 primer design assistance softwarewas used (The development of Primer3 and the Primer3 web site was fundedby Howard Hughes Medical Institute and by the National Institutes ofHealth, National Human Genome Research Institute. Under grantsR01-HG00257 (to David C. Page) and P50-HG00098 (to Eric S. Lander):http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Afluorescently labeled probe was synthesized from the designed probesequence. The 5′ end was labeled with FAM and the 3′ end was labeledwith TAMRA as fluorescent labels. Using these probes, real-time PCRconditions were established for the TaqMan procedure using the standardplasmid as a template. After optimal conditions were determined, acomparison of various primer (see Table 10) and probe combinations wasperformed. Satisfactory amplification was obtained with each of thecombinations, and the detection efficiency was approximately the same.

Table 11 shows the sequences of the designed probes. FIG. 4 shows theresults obtained using each type of primer.

TABLE 11 Probe name Sequence PRP-Taq1 5′-TCGACCCCGTCCGGAGCCAC-3′(SEQ ID NO: 9) PRP-Taq2 5′-AGCTGAAGGAGGAGATCGACCCC-3′ (SEQ ID NO: 10)

Example 5 Establishment of Real-Time PCR Conditions

By following the real-time PCT procedure in Example 1 and using theprobes described in Example 4, the parameters of primer concentration,probe concentration, PCR reaction temperature, and time were varied todiscover the optimal conditions. Tables 12 and 13 show the selectedconditions.

TABLE 12 Conc. μL Premix  2x 10 ROX 50x 0.4 Probe 10 μM 0.8 Primer pair 5 μM each 0.8 H₂O 4.0 Total 16.0

TABLE 13 Step Temp. Time Cycle Ramp time 1 50° C.  2 min 1 Auto 2 95° C.10 min 1 Auto 3 95° C.  5 sec 40 Auto 60° C. 30 sec Auto 4 20° C.  1 min1 Auto

Example 6 Verification of Universality in Wheat Varieties

A comparison of the detected amount was performed by real-time PCR usingthe TaqMan procedure for 40 varieties of wheat. The amplificationproduct Wx012 (SEQ ID NO: 31) amplified by the primers Wx012-F andWx012-R was used as a comparative control. The PRP03-F and PRP03-R wereused as the primers and PRPTaq-1 was used as the probe. Theamplification product PRP03 (SEQ ID NO: 15) amplified by the primersPRP03-F and PRP03-R was detected in all 40 varieties. Wx012 was detectedin all varieties except durum wheat and waxy wheat varieties. In thecomparison with PRP03, the Wx012:PRP03 ratio was approximately 1:1 inall varieties wherein Wx012 was detected.

FIG. 5 shows the detection examples of 20 of the 40 varieties of wheatwherein PRP03 was detected. In FIG. 5 the terms Hatsumochi andMochiotome indicate varieties of waxy wheat, and AC-Navi refers to theAC Navigator variety of durum wheat. These were all detected.

Example 7 Estimation Test of Contamination Rate by Hypothetical GM Wheat

At the present time GM wheat is produced by Monsanto, etc., but it isvery difficult to obtain. Therefore, a test was conducted using ahypothetical GM wheat to verify whether or not an estimation of the GMwheat contamination rate is possible by quantitation of the endogenouswheat gene discovered by the present invention.

Because the GM wheat produced by Monsanto is imparted with a Roundup®resistance gene, it was decided to use GM soybeans, which are a GMOimparted with the same Roundup® resistance gene. In other words, GMsoybean seeds were pulverized, AC Barrie variety wheat seeds werepulverized and mixed therewith, and that was used as the hypothetical GMwheat. The mix ratio of both was investigated in preliminary tests sothat the internal standard ratio would approach 1, and the mix ratio ofGM soybeans to wheat was determined as 6:94. Quantitation of theRoundup® resistance gene and the endogenous gene (PRP region) wasperformed by real-time PCR with a TaqMan probe using DNA extracted fromthe hypothetical GM wheat as a template under the conditions shown inExample 5. (PRP-Taq2 was used as the probe, and the primers shown inTable 14 were used as primers. In the table, RRS refers to the Roundup®resistance gene.) The sequence amplified at that time is identified asSEQ ID NO: 32. The numerical value resulting when the obtained RRSquantitative value was divided by the endogenous wheat gene quantitativevalue was used as an internal standard ratio (1.0).

TABLE 14 Amplicon size Gene Name Sequence (bp) PRP prp03-F5′-aag cca ccg atg act gac aat-3′ 231 (SEQ ID NO: 3) prp07-R5′-cgc aaa tga taa tta cag aat agt agt ac-3′ (SEQ ID NO: 4) RRS RRS2-F5′-cct tta gga ttt cag cat cag tgg-3′ 121 (SEQ ID NO: 33) RRS2-R5′-gac ttg tcg ccg gga atg-3′ (SEQ ID NO: 34)

DNA was extracted from a sample obtained by arbitrarily mixing thehypothetical GM wheat with separately pulverized wheat (Yecora Rojovariety), and using this DNA as a template and following the method ofthe present invention, the endogenous wheat gene and Roundup® resistancegene were quantitated. FIG. 6 shows the hypothetical GM wheatcontamination rate determined from those results. It was confirmed thatthe contamination rate determined by a calculation in accordance withthe method of the present invention shows a high correlation with theactual mix ratio. More specifically the detected amount of GM wheat(copy number concentration: copy/μL) per volume of obtained templatesolution was divided by the detected amount (copy number concentration:copy/μL) per volume of obtained template solution of endogenous wheatgene. That quotient was divided by the internal standard ratio of 1.0and multiplied by 100, and the resulting quotient was used as the GMcontamination rate.

Example 8 Specificity Verification Test

A specificity test was performed to verify that the method according tothe present invention does not have cross-reactivity with other crops.Barley, oats, rye, rice, sorghum, rapeseed, maize, buckwheat, and kibimillet were used as the other crops. DNA was extracted from these crops,and real-time PCR was performed using the DNA as a template under theconditions established in accordance with the method presented inExample 5.

Two types of primer pairs, PRP03-F and PRP03-R, and PRP03-F and PRP07-Rwere used, and PRP-Taq2 was used as the probe.

FIG. 7 shows the results. When the primer pair of PRP03-F and PRP07-Rwas used, 140 copies per 1 μg of DNA were detected in wheat, but in theabove crops no more than 0.2 copies were detected. In other words, thenonspecific detection rate in those crops was no more than 0.05%, andbecause this value is much lower than the standard error in wheatdetection, it was confirmed that these crops do not affect thequantitation of the endogenous wheat gene. When the primer pair ofPRP04-F and PRP06-R was used, although a detection of 0.3% with respectto wheat was found in barley, the rate was no more than 0.05% in othercrops. Therefore, even when this primer pair was used, the value wasmuch lower than the standard error in wheat detection, and it wasconfirmed that these crops do not affect the quantitation of theendogenous wheat gene.

1.-13. (canceled)
 14. A kit for detecting or quantitating an endogenouswheat DNA sequence in a test sample by the polymerase chain reaction,comprising at least one primer pair selected from (i) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 3 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 4; (ii) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 5 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 4; (iii) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 3 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 6; (iv) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 5 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 6; (v) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 3 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 7; (vi) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 3 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 8; (vii) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 5 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 8; (viii) a primer pairconsisting of a nucleic acid molecule comprising the nucleotide sequenceidentified as SEQ ID NO: 5 and a nucleic acid molecule comprising thenucleotide sequence identified as SEQ ID NO: 7; and (ix) a primer pairconsisting of a pair of nucleic acid molecules, wherein one nucleic acidmolecule in the primer pair in (ix) comprises a nucleotide sequence incommon with at least 80% continuous nucleotide sequence of one nucleicacid molecule in one of the primer pairs in (i) to (viii) above, and theother nucleic acid molecule in the primer pair in (ix) comprises anucleotide sequence in common with at least 80% continuous nucleotidesequence of the other nucleic acid molecule in the same primer pair in(i) to (viii) above.